The invention relates to the fields of medicine and diagnostics. More in particular, the invention relates to medicine and the diagnosis of tumours.
Recurrent translocations acquired in a process of transformation are well recognised in nodal B-cell lymphomas. These translocations characterise distinct subtypes of disease and involve genes controlling cell proliferation and apoptosis. BCL2, which suppresses apoptosis, was cloned from the t(14;18)(q21;q32) found in most cases of follicular B-cell lymphoma, whereas translocations involving the BCL1/CyclinD1 gene on chromosome 11q13 are seen in nearly all cases of mantle cell lymphoma.1 
By contrast, the genetic mechanisms underlying the genesis and disease progression of extranodal marginal zone B-cell lymphomas of the mucosa-associated lymphoid tissue (MALT) type, a recently recognised distinct subtype of B-cell Non-Hodgkin""s Lymphoma""s (NHL), are not known.2 MALT lymphomas account for five to ten percent of all NHLs and the vast majority of lymphomas arising at extranodal sites. They originate in a setting of chronic inflammation triggered by chronic infection or autoimmune disorders, such as Helicobacter pylori gastritis, Sjxc3x6gren""s syndrome, and Hashimoto""s thyreoiditis.3 In vitro experiments have shown that H. pylori specific T-cells provide contact dependent help for the growth of the malignant B-cells of gastric MALT.4 The etiological link between low-grade gastric MALT lymphomas and H. pylori infection has also been demonstrated by the regression of some cases with antibiotic therapy.5,6 The preferential use of immunoglobulin variable region genes (VH) associated with autoimmune disorders indicate that some MALT lymphomas may arise from autoreactive B-cells.7,8 
Since biopsies of these lymphomas are relatively rarely subjected to cytogenetic analysis and their in vitro proliferation is often poor, abnormal karyotypic data have been published for only 46 low-grade MALT lymphomas9-17, 5 extranodal small lymphocytic lymphomas of probable marginal zone origin 18-20, and 23 high-grade gastric MALT lymphomas14,15. Recurrent abnormalities in these cases include trisomies of chromosomes 3, 7, 12, and 1811,17,21, the t(1;14)(p22;q32) which has been described in two cases 17 and the t(11;18)(q21;q21). The t(11;18)(q21;q21) has been detected in 15 out of the 51 low-grade lymphomas arising from various extranodal sites9,12,14,18-20 but in none of the high-grade MALT lymphomas or any other subtype of NHL. In the largest cytogenetic series, this translocation has been found in 7 out of 13 cases of low-grade MALT lymphomas with an abnormal karyotype.14 These data clearly indicate that the t(11;18) represents the most frequent structural abnormality in low-grade MALT lymphomas and seems to specifically characterise this disease entity. An attempt to delineate the breakpoint at 18q21.1 has been described by Akagi et al. (Genes, Chromosomes and Cancer, 24 (1999): 315-321). A detailed characterization, however, is urgently needed.
In one aspect, the present invention provides a detailed molecular genetic characterisation of the 11q21 and 18q21 breakpoint regions in MALT lymphomas characterised by the t(11;18)(q21;q21). The invention further identifies that the API2 gene also known as c-IAP222, HIAP123 and MIHC24, an inhibitor of apoptosis, and a novel gene on 18q21, named MLT, are rearranged in this translocation. The invention also identifies that truncation of the API2 gene distal to its three BIR domains and fusion of this truncated gene with the carboxy-terminal region of MLT may lead to increased inhibition of apoptosis and thereby confer a survival benefit to MALT type B-cell lymphomas. In other words, the present invention discloses that, surprisingly, truncation of the API2 gene and fusion to the new MLT gene is crucial for the development of MALT type B-cell lymphomas.
In one aspect, the invention teaches that the t(11;18)(q21;q21) associated with extranodal marginal zone B-cell lymphomas of the MALT type results in the expression of a chimeric transcript fusing 5xe2x80x2-API2 on chromosome 11 to 3xe2x80x2-MLT on chromosome 18. The occurrence of the t(11;18) translocation in not less than 17 out of 51 published cases of low-grade MALT lymphomas9,12,14,18-20 including the two cases described herein, along with its presence as the sole cytogenetic abnormality in 16 out of the 17 reported cases indicates that the t(11;18) represents one of the main recurrent, disease-specific translocations in NHL.
Several observations point to the API2-MLT fusion as the oncogenic lesion underlying the t(11;18). The chimeric cDNA was cloned from two independent tumours. In one case, the genomic breakpoints were also cloned and the structure of both genes and the localisation of the breakpoints is in agreement with the expression of the fusion transcript. The cryptic deletion of the 3xe2x80x2 part of API-2 in case 1 precludes the expression of a reciprocal MLT-API2 transcript in this case. As a result of the deletion, the 5xe2x80x2-end of the MLT gene is fused to the 5xe2x80x2-end of the MMP20 gene on the der(18). As both genes are present on opposite strands of the DNA no MLT-MMP20 transcript is expressed. Furthermore, FISH experiments with PACs for respectively MLT, API2 and MMP20 clearly suggest that in case 2 a balanced translocation occurred not involving a break in the MMP20 gene further arguing against any significance of the MLT-MMP20 fusion.
API2 belongs to the family of inhibitor of apoptosis proteins (xe2x80x9cIAPxe2x80x9d), which play an evolutionary conserved role in regulating programmed cell death in diverse species (International Patent Application WO 97/06182 to Rothe and Goeddel). The IAP genes were first identified in baculoviruses in which they demonstrated an ability to suppress the host cell apoptotic response to viral infection.33 Subsequently, five human IAP relatives have been described: NIAP, API1 (also known as cIAP1, HIAP2, MIHB), API2 (cIAP2, HIAP1, MIHC), XMAP-hILP and survivin.22-24,34-37 The common structural features of all IAP family members is a motif termed baculovirus IAP repeat (xe2x80x9cBIRxe2x80x9d) occurring in one to three copies, a caspase recruitment domain or CARD30 located between the BIR domain(s) and a carboxy-terminal zinc binding RING finger domain3 that is present in all IAPs with the exception of NIAP and survivin. The human API1 and API2 proteins were originally identified as proteins that are recruited to the cytosolic domain of the tumour necrosis factor (TNF) receptor II via their association with the TNF-associated factor (TRAF) proteins, TRAF-1 and TRAF-222 and have been subsequently shown to suppress different apoptotic pathways by inhibiting distinct caspases, such as caspase-3, caspase-7, and pro-caspase-9.35-38 
The function of the novel MLT gene (also known as xe2x80x9cMALT1xe2x80x9d) located on chromosome 18q21 is not yet known. Its closest homologue is a hypothetical C. elegans gene. The carboxy-terminal part of this gene is characterised by the presence two Ig-like C2-type domains and a domain similar to the murine Ig gamma chain VDJ4 sequence (Accession no. M13070). The C2 domains are only present in the longer fusion cDNA of case 2 and thus probably have no functional significance in the tumour.
The molecular mechanism of action of the API2-MLT fusion remains to be elucidated. Without being limited by theory, it is hypothesised that the fusion protein resulting from the t(11;18) may lead to increased inhibition of apoptosis and thereby confer a survival advantage to MALT lymphomas and allow antigen-independent proliferation. Indeed, MALT lymphomas have been shown to display low levels of apoptosis39, to escape from FAS-mediated apoptosis40 and about 20% of low-grade MALT lymphomas do not respond to H. pylori eradication therapy.6 The truncation of API2 after the BIR domains could release their anti-apototic effects from regulation by the CARD and RING domains. Recent studies have shown that the BIR domain-containing regions of API1 and API2 are sufficient for inhibition of caspases and suppression of apoptosis.35 The BIR domains of one of the Drosophila homologues (xe2x80x9cDIAP1xe2x80x9d) were demonstrated to suppress apoptosis in the Drosophila eye disk, whereas the full-length protein exhibited less activity. Moreover, transgenic flies over-expressing the RING domain alone exhibited increased cell death in the eye, suggesting that the RING domain may act as a negative regulator of cell death suppression in some instances.34 On the other hand, a specific role for the carboxy-terminal MLT domain is suggested by its consistent presence in the fusion and by the recurrency of the t(11;18) in MALT-lymphoma. This is supported by the observation that full-length API1 and API2 were somewhat more potent in caspase inactivation than constructs lacking the RING domain.35 It is possible that the presence of the MLT domain would stabilise the fusion protein, increase its affinity for protein interaction or influence its subcellular localisation thereby modulating its interactions with other proteins.
The mechanism of gene deregulation by the t(11;18) differs from that seen in most of the B-cell lymphoma-associated translocations, which involve one of the immunoglobulin loci on 14q32, 2p12, or 22q11 and lead to deregulated expression of the incoming oncogene due to the proximity of potent B-cell transcriptional enhancers within the immunoglobulin loci.41 In this case, the expression of the fusion gene is driven from the promoter of its 5xe2x80x2 partner, API2. This agrees with the observation that API2 mRNA is highly expressed in adult lymphoid tissues, including spleen, thymus, and peripheral blood lymphocytes and also in fetal lung and kidney is in agreement.23 It is also interesting to note that the IAP family member survivin is strongly expressed in apoptosis-regulated human fetal tissues, but not in terminally differentiated adult tissues.42 Survivin becomes prominently expressed in transformed cell lines and in most human cancers. Survivin expression was also found in 50% of high-grade NHL (centroblastic, immunoblastic), but not in low-grade lymphomas (lymphocytic).37 
At the genomic level, the rearrangements appear to be heterogeneous. The breakpoint in MLT occurred in two different introns for both cases. In the API2 gene the breakpoint occurred in the same intron for both cases but it was associated with the deletion of the 3xe2x80x2-end of the gene in only one tumour. The cytogenetic analysis of MALT lymphoma is often hampered by their poor proliferation in vitro. However, the physical maps and the genomic clones that the present invention teaches allows the development of a wide variety of sensitive detection methods for this rearrangement, such as but not limited to interphase FISH assays and assays based on the specific amplification of nucleic acid encompassing the t{11:18} breakpoint. Alternatively, the fusion mRNA or the fusion protein provide new molecular targets for diagnosis.
In one aspect, the invention provides a method for determining whether a tissue sample or an analogue and/or derivative thereof comprises a cell with a chromosome {11:18} translocation associated with malignancies such as mucosa-associated lymphoid tissue (MALT) lymphoma""s the method comprising subjecting nucleic acid from the sample to an amplification reaction using a primer that is complementary to a nucleic acid sequence which in humans lies on chromosome 11 region q21-22.3 and a primer that is complementary to a nucleic acid sequence which in humans lies on chromosome 18 region q21.1-22, and determining the presence of any amplified product. Preferably, the tissue sample is taken from a human individual. Preferably, the individual is suffering from or at risk of suffering from a disease.
The nucleic acid may comprise chromosomal DNA, RNA or any other type of nucleic acid. With the knowledge of the region in which the {11:18} translocation occurs, a person skilled in the art is capable of developing specific nucleic acid amplification methods that allow the unambiguous detection of the translocation in a tissue sample. Such assays may be developed based on nucleic acid sequence information presented here. However, it is clear that using the teachings of the present invention, i.e., the identification of the break point and the methods and means to do so, additional nucleic acid sequence information on the region surrounding the break point can be obtained and that the use of such sequence information for the development of detection assays for the translocation falls within the scope of the present invention. With a xe2x80x9ccomplementary primerxe2x80x9d is meant a primer that can hybridise to another sequence under relatively mild hybridisation conditions, i.e., hybridisation conditions that allow nucleic acids with sequences with some mismatches to hybridise to each other. The number of mismatches among others determines the specificity and the efficiency of polymerisation. A complementary primer should comprise not more than 30% mismatches, preferably not more than 20% more preferably not more than 10%. Preferably, the primer does not comprise mismatches.
Amplification methods that may be used in a method of the invention include but are not limited to polymerase chain reaction, NASBA and intracellular PCR.